Eukaryotic cell division proceeds through a highly regulated cell cycle comprising consecutive phases beginning with a phase termed G1, and followed by phases termed S (DNA synthesis), G2 and M (Mitosis). Disruption of the cell cycle or cell cycle control can result in cellular abnormalities or disease states such as cancer, which arise from multiple genetic changes that transform growth-limited cells to highly invasive cells that are unresponsive to normal control of growth. Transition of normal cells to cancer cells can arise through loss of correct function in DNA replication and DNA repair mechanisms. Normal dividing cells are subject to a number of control mechanisms, known as cell-cycle checkpoints, which maintain genomic integrity by arresting or inducing destruction of aberrant cells. Investigation of cell cycle progression and control is consequently of significant interest in designing anticancer drugs (Flatt, P. M. and Pietenpol, J. A. Drug Metab. Rev., (2000), 32(3-4), 283-305; Buolamwini, J. K. Current Pharmaceutical Design, (2000), 6, 379-392).
Cell cycle progression is tightly regulated by defined temporal and spatial expression, localization and destruction of a number of cell cycle regulators, which exhibit highly dynamic behaviour during the cell cycle (Pines, J., Nature Cell Biology, (1999), 1, E73-E79). For example, at specific cell cycle stages some proteins translocate from the nucleus to the cytoplasm, or vice versa, and some are rapidly degraded (Kohn, Molecular Biology of the Cell (1999), 10, 2703-2734).
Many cancer cells carry abnormalities in G1 checkpoint-related proteins such as p53, Rb, MDM-2, p16 INK4 and p19 ARF (Levine (1997) Cell, 88:323). Alternatively, mutations can cause overexpression and/or over-activation of oncogene products, e.g., Ras, MDM-2 and cyclin D, which reduce the stringency of G1 checkpoint. In addition to these mutations, excessive growth factor signaling can be caused by the overexpression of growth factors and can reduce the stringency of G1 checkpoint. Together with loss-of-function and gain-of-function mutations, continuous activation by growth factor receptors or downstream signal-transducing molecules can cause cell transformation by overriding the G1 checkpoint. A disrupted or abrogated G1 checkpoint contributes to higher mutation rates and the many mutations observed in cancer cells. As a result, many cancer cells depend on G2 checkpoint for survival against excessive DNA damage (O'Connor and Fan (1996) Prog. Cell Cycle Res., 2:165).
The G2 cell cycle checkpoint restricts the onset of mitosis until DNA replication and repair are complete. Malfunction of the G2 checkpoint would allow premature onset of mitosis prior to the completion of DNA replication and repair, producing daughter cells lacking a substantial portion of the genomic DNA or harboring mutations. Functions of the G2 checkpoint includes detecting DNA damage and generation of signal that can lead to cell cycle arrest when DNA damage is detected. The mechanism that promotes the cell cycle G2 arrest after DNA damage is believed to be conserved among species from yeast to human.
Kinases play a central role in cell cycle regulation. Defects in various components of signal transduction pathways have been found to account for a vast number of diseases, including numerous forms of cancer, inflammatory disorders, metabolic disorders, vascular and neuronal diseases (Gaestel et al. Current Medicinal Chemistry (2007) 14:2214-2234). In recent years, kinases that are associated with oncogenic signaling pathways have emerged as important drug targets in the treatment of various diseases including many types of cancers.
The mammalian target of rapamycin (mTOR), also known as mechanistic target of rapamycin, is a serine/threonine protein kinase that regulates cell growth, translational control, angiogenesis and/or cell survival. mTOR is encoded by the FK506 binding protein 12-rapamycin associated protein 1 (FRAP1) gene. mTOR is the catalytic subunit of two complexes, mTORC1 and mTORC2. mTORC1 is composed of mTOR, regulatory associated protein of mTOR (Raptor), mammalian LST8/G-protein β-subunit like protein (mLST8/GβL), PRAS40, and DEPTOR. mTOR Complex 2 (mTORC2) is composed of mTOR, rapamycin-insensitive companion of mTOR (Rictor), GβL, and mammalian stress-activated protein kinase interacting protein 1 (mSIN1).
Apart from their subunits, mTORC1 and mTORC2 are distinguished by their differential sensitivities to rapamycin and its analogs (also known as rapalogs). Rapamycin binds to and allosterically inhibits mTORC1, but mTORC2 is generally rapamycin-insensitive. As a result of this rapamycin-insensitive mTOR signaling mediated by mTORC2, cancer cells treated with rapamycin analogs usually display only partial inhibition of mTOR signaling, which can lead to enhanced survival and resistance to rapamycin treatment. Typically, mTOR inhibitors suppress cell-cycle progression in the G1 phase.
Paclitaxel is a cytotoxic chemotherapeutic used as an anti-tumor agent in the treatment of carcinomas of the ovary, breast, lung and in the treatment AIDS related Karposi's sarcoma. Originally derived from the western yew, Taxus brevifolia, paclitaxel has been used to treat breast cancer by pre-operatively administering the drug systemically. At the molecular level, paclitaxel exerts an antitumor activity through its ability to promote apoptosis (programmed cell death) by inducing the assembly of microtubules from tubulin dimers and preventing microtubules from depolymerization. The stabilized microtubules inhibit normal dynamic reorganization of the microtubule network that is essential for vital interphase and mitotic functions. In addition paclitaxel induces abnormal arrays or “bundles” of microtubules throughout the cell cycle and multiple asters of microtubules during mitosis. As a result, paclitaxel increases the fraction of cells in G2 or M phase.
Although impressive success has been achieved using this approach, some tumors either do not respond or become resistant to treatment with paclitaxel. Moreover, a significant number of cases do not result in a clinically satisfactory outcome either because the tumors are not reduced or because the side effects require that paclitaxel dosing be discontinued.